Leaf proteins are potentially the cheapest and most abundant source of protein in the world (Wikipedia, 2008a, Pirie, 1987). (Full reference citations of these and all references mentioned herein are included at the end of the specification.) They are also highly nutritious and have many desirable functional characteristics which could make them useful in both food and industrial products.
The term “leaf protein” as used in this invention disclosure is intended to refer to all water-soluble proteins contained in plant leaves. It is well known that soluble leaf proteins are found in all known chlorophyll-containing plants. The present invention pertains specifically to soluble leaf proteins.
Approximately half of the soluble protein in plant leaves is made up of “rubisco” (ribulose-1,5-bisphosphate (RUBP) carboxylase/oxygenase or “RuBisCO”) (Johal, 1982). Rubisco, which is found in all known green plants, appears to be the most abundant leaf protein, and it may be the most abundant protein on earth (Wikipedia, 2008b). Rubisco is the enzyme which catalyzes both the carboxylation and oxygenation of RUBP in plants, i.e., the key reactions in photosynthesis and photorespiration (Tso, 1990). Rubisco is the primary component of “fraction-1 protein,” a term developed by Wildman (1983) to refer to the portion of the soluble leaf protein which can be crystallized out during leaf protein processing.
Rubisco has nutritional value comparable to casein, the milk protein (Wildman, 1983). Studies have shown that rubisco has a significantly higher Protein Efficiency Ratio (PER, i.e., weight gained/protein consumed) than either casein or egg protein (Tso, 2006). Tornatzky (et al., 1996) reported that rubisco appears suitable for kidney dialysis patients and other persons whose bodies do not produce adequate protein, due in part to the fact that rubisco crystals can be washed clean of salts (Tornatzky et al., 1996).
Rubisco also has excellent binding, gelling, foaming, whipping and emulsifying characteristics (Wildman, 1983; Sheen 1991). In addition, rubisco is colorless, tasteless and odorless, which makes it attractive for incorporation into food or industrial products. (Wildman, 1983). Rubisco is relatively stable and can be shipped in crystalline form or produced in a powder (Tornatzky et al., 1996). Given these desirable nutritional and functional properties, rubisco may prove suitable for incorporation into a range of both food and non-food products for such purposes as a nutritional supplement, binding agent or emulsifier. In fact, Wildman (1983) wrote that the functional properties of rubisco are similar to egg albumin or casein.
The remaining half of soluble leaf proteins do not crystallize as readily as fraction-1 proteins. They are sometimes referred to as “fraction-2” proteins (Wildman, 1983), but a term used to describe those soluble proteins which do not crystallize during leaf protein processing. These proteins share, however, many of the same beneficial traits as rubisco. They have a PER and nutritional quality comparable with casein (Tso, 2006; Wildman, 1983). Like rubisco, they are colorless and tasteless (Tso, 2006). They are also water-soluble (Wildman, 1983). With appropriate extraction methods, fraction-2 proteins could demonstrate the same functional properties as rubisco and have the same commercial applications (Wildman, 1983). Both the so-called “fraction-1” and “fraction-2” proteins are pigment-bound proteins.
Potential yields of leaf proteins are very large. Wildman (1983) reported yields of 6% of total plant solid dry matter as leaf proteins in tobacco. In our own field trials using tobacco as a source of leaf protein, we have obtained crude protein yields of approximately 13% of total leaf dry weight from tobacco variety MD 609 LA, a variety of Maryland tobacco which contains low alkaloids. Our average annual biomass yields exceed six dry tons per acre, when we cultivate tobacco for leaf protein recovery. Since the leaves represent about half of this total biomass, our projected leaf protein yields are approximately 800 pounds/acre.
Pirie may have been the first to seriously propose the use of leaf proteins as a source of nutrition, during the 1940's (Pirie, 1942, 1987). However, the protein extraction methods developed by Pirie resulted in green protein preparations which had odor, taste and texture which rendered them undesirable for human consumption (presumably due to inadequate removal of chlorophylls) (Wildman, 1983).
During the 1970's researchers at the USDA Western Regional Research Center modified and improved the technology developed by Pirie to produce a leaf protein concentrate from alfalfa which they called “Pro-Xan.” In the later versions of this process, soluble proteins were coagulated by heat to produce a bland, off-white material containing 90% protein (Wildman, 1983). Wildman (1983) reported that the rubisco in this resulting protein was denatured together with the fraction-2 protein, and that it lost its functional characteristics.
In the early 1980's Wildman (1983) and Wildman and Kwanyuen (1981) developed a technology for extracting rubisco from tobacco leaves which relied on application of sodium metabisulfite. The aerial portions of freshly harvested tobacco plants were sprayed with a 0.5 percent solution of sodium metabisulfite as they fell into a pulping device. The pulp was placed into a screwpress which led to a recovery of a “green juice” containing the soluble proteins. This green juice was pumped into a heat exchanger which rapidly heated the juice to about 125° F. The juice was then rapidly cooled to room temperature. Wildman wrote that this abrupt temperature change aided in particulate matter aggregation to which green pigments and other lipoidal compounds are attached. The green juice then entered a continuous flow centrifuge which removed the starch and 85% of the green particulate material. A partially clarified brown containing the soluble proteins juice emerged from the centrifuge. The partially clarified brown juice was then pumped to a rotary vacuum filter which removed the last traces of green sludge. The clear brown juice emerging from the filter was then sent to a storage tank, where crystallization of the rubisco protein began within 3-6 hours of storage and was completed within 3-4 hours after crystallization began. Wildman reported that he added acid to the mother liquid at pH 4 in order to precipitate the fraction-2 proteins, while noting that this was not a desirable method because it resulted in denaturation of the proteins.
Bourque (1982) developed a method for the crystallization of rubisco which involved mixing a fraction-1 protein solution with a precipitant solution with a pH generally in the range of 4.8-7.2, to produce a mixed solution of protein solution mixed with precipitant solution having a final pH in the range of pH 6.6-7.0, which lead to crystallization of the fraction-1 protein.
Bourque mentioned that the protein solution could be obtained by standard fraction-1 plant protein purification processes previously described in the literature. For example, he wrote that it is possible to homogenize the leaves in a high salt (i.e., 1.0 M NaCl) buffer, filter the homogenate to remove larger solids, and then centrifuge to obtain a supernatant containing soluble protein, and exchanging a low salt buffer (i.e., 0.2 M NaCl) for the high salt buffer, and then eluting the protein solution from a column with the low salt buffer, and then precipitating the protein by adding a precipitant such as ammonium sulfate to provide 30-50% precipitant saturation. The resulting precipitate could then be crystallized using the technique disclosed by Bourque. Bourque reported that his invention was suitable for use with fresh leaves from tobacco, corn, potato, tomato or alfalfa.
Johal (1982, 1983 and 1986) developed a technique for the crystallization of rubisco to over 90% purity. In this technique, the leaves would first be homogenized in a buffer solution containing tris-HCL or phosphate or borate, at concentrations of 0.05M to 0.2M. The buffer solution would have a pH range of 7.8 to 8.25, with the optimal pH around 8.2. Polyethylene glycol would be added to represent up to 15% of the solution. The supernatant would be refrigerated at about 4° C. Johal (1982) reported that adding magnesium chloride would increase crystal yield. Johal reported that his technique had proven successful using several species, including tobacco, alfalfa, ryegrass, tomato and spinach.
DeJong (1982) reported a coagulation method for preparing leaf protein concentrates. Physically macerated leaves were subject to a cross-linking reagent such as gluteraldehyde, and then treated with potassium bisulfate to lower the pH to between 3 and 5. This treatment would precipitate soluble protein from the green juice, and then coagulate suspended proteins, resulting in the separation of a leaf protein concentrate.